Methods of purifying synthetic oligonucleotides

Methods of Purifying Synthetic Oligonucleotides

From the pioneering work of Letsinger and Lunsford to today’s applications in synthetic biology, where one can synthesize entire chromosomes (by PCR /overlapping oligos methodologies) there are a number of purification methods including but not limited to;

1. RP-HPLC. One uses the Trityl on strategy, where the trityl group is left after the last addition cycle, this provides enough hydrophobicity to be retained in a C-18 column, and separation is possible. This works up to fragments of about 30-35 nucleotides in length, after that , the overall hydrophobicity of the oligos does not allow separation

2. Variations of RH-HPLC, this include the so called cartridge based purifications; using trityl on, one uses small 1-2 cm in length so called Sep-Paks(Waters Corp) to purify; generally this renders purities of 80-90 %, and again limited to 30-35 mers

3. Capillary Electrophoresis (CE). Unless one can collect the eluates and very small amounts of oligo are needed, this is not truly a purification method, rather a QC method to check purity.

4. Ion-exchange HPLC. This method can purify with better resolution, in some cases, than RP-HPLC; makes use of ion-pairing agents. The drawback here is the potential for HPLC damage if leftover salts are left in the HPLC system

5. Polyacrylamide Gel Electrophoresis (PAGE). This is perhaps

6. The oldest method, less automated one (does not require expensive equipment) and after all these years in our opinion, remains perhaps the best purification method for oligonucleotides, due to the exquisite single base resolution properties. The drawback is that one has to manually prepare the GEL (10-20%) and works well to purify oligos in the range of 50-100 OD’s.